The invention relates to processes for the quantiative optical analysis of the intracellular concentration of cyclic guanosine 3′,5′-monophosphate (CGMP) or cyclic adenosine 3′,5′-monophosphate (cAMP). These processes may be useful for identifying and evaluating the pharmacological properties of test substances which influence the activity of receptors or enzymes involved in the synthesis or degradation of the cyclic nucleotides cGMP and cAMP. As a result, the process is suitable, for example, for finding modulators of soluble and membrane-bound guanylate cyclases, phosphodiesterases (PDEs), NO synthases and G-protein-coupled receptors (GPCRs).
The invention describes a process in which the intracellular concentrations of cGMP or cAMP are measured with the aid of recombinant cell lines. These cell lines express recombinantly a combination of particular ion channels activated by cyclic nucleotides (CNG channels), a calcium-sensitive photoprotein and a receptor or an enzyme for which modulators are to be found. The process is suitable for automation and for high-throughput screening (HTS) and ultra high-throughput screening (uHTS) for modulators.
Current processes for measuring the intracellular concentration of cGMP or cAMP have the disadvantage of being very expensive, due to the use of radioactivity, being only partially automatable, if at all, and being very complicated.
Conventional commercial systems for determining cGMP and cAMP are radioactive techniques such as the radioimmunoassay (RIA; e.g. from IBL, Hamburg, Germany), the scintillation proximity assay (SPA; Amersham Bioscience, UK) and the non-radioactive enzymed-linked immunosorbent assay (ELISA; for example from Amersham Bioscience, UK).
The above-described processes cannot be used for measuring the intracellular cGMP and cAMP concentrations in living cells “in situ”. To carry out the measurements, the cells need to be disrupted and the cyclic nucleotides then be extracted.
Another process for measuring intracellular cAMP is the measurement with the aid of inducible promoter systems (“cAMP response element”, CRE) and the enzyme luciferase (Goetz et al., J. Biomol. Screen. (2000) 5 377-384). The process has the disadvantage, however, that both transcription and translation need to take place in order for the signal to form, causing the measurement to last for hours. This method is thus not suitable for rapid measurement of the actual cAMP concentrations in cells.